Process for the preparation of pepsin inhibitors

ABSTRACT

New substances named &#39;&#39;&#39;&#39;substance S-PI&#39;&#39;&#39;&#39; and &#39;&#39;&#39;&#39;substance Me SPI&#39;&#39;&#39;&#39; are concerned which are effective as pepsin inhibitor, the latter substance being methyl ester of the former. The substance S-PI is prepared by fermentation of a new strain of microorganism which has been isolated from soil by the present inventors and named the Streptomyces naniwaensis (EF 44-201), whereas the substance Me S-PI is prepared by methyl esterfying the former substance. A specimen of the Streptomyces naniwaensis (EF 44-201) has been deposited to the American Type Culture Collection under the assigned Number ATCC 21689.

United States Patent Murao et al.

[11] 3,819,486 [4 June 25,1974

[ PROCESS FOR THE PREPARATION OF PEPSIN INHIBITORS [73] Assignee: EisaiCo., Ltd., Tokyo, Japan [22] Filed: Jan, 12,1973

[21] Appl. No.: 323,243

Related US. Application Data [62] Division of Ser. No. 138,083, April28, 1971.

[30] Foreign Application Priority Data Apr. 29, 1970 Japan 45-35900 [52]US. Cl. 195/80 R, 260/112.5 [51] Int. Cl Cl2d 13/06 [58] Field of Search195/80 R [56] References Cited OTHER PUBLICATIONS Umezawa et al.,Journal of Antibiotics, 23,

Morishima et al., Journal of Antibiotics, 23, 263265 (1970).

Primary Examiner-Lionel M. Shapiro Attorney, Agent, or Firm-Cooper,Dunham, Clark, Griffin & Moran ABSTRACT New substances named substance8-H and substance Me S-PI are concerned which are effective as pepsininhibitor, the latter substance being methyl ester of the former. Thesubstance S-Pl is prepared by fermentation of a new strain ofmicroorganism which has been isolated from soil by'the present inventorsand named the Streptomyces naniwaensis (EF 44-201), whereas thesubstance Me S-PI is prepared by methyl esterfying the former substance.A specimen of the Streptomyces naniwaensis (EF 44-201) has beendeposited to the American Type Culture Collection under the assignedNumber ATCC 21689.

fz ausianla n ri re PROCESS FOR THE PREPARATION OF PEPSIN INHIBITORSThis is a division, of application Ser. No. 138,083

The substance 8-H and the substance Me S-PI according to the presentinvention are represented by the formula CH; CH: C53 /CH3 CH3 /CH:| (3HCH CH CH2 OH wherein R is hydrogen or methyl, and when R is hydrogen,-the formula represents the substance S-PI, whereas when R is methyl, theformula represents the substance Me S-PI.

According to the present invention, the substance S-Pl is obtained byfermenting the Streptomyces naniwaensis (EF 4420l in a suitable nutrientmedium and recovering the said substance as a metabolite from thefermentation broth. The other substance, that is, the substance Me SPI,on the other hand, is obtained by further treatment of the saidsubstance S-PI with a methylating agent.

i It was well known that substances having inhibitory effect againstenzymatic action of trypsin and microbial proteases are widelydistributed in natural sources, such as beans, cereals, potatoes,egg-white, blood and the like (comp. The Enzymes 4, pages 128 and 213(I960), reported by P. D. Boyer et al).

It was, however, hitherto scarcely known that a substance havinginhibitory effect against protease and especially acid protease isformed as a metabolite of mic'roorganism.

K. Matsuhima et al., reported that they had succeeded to isolate aprotease inhibitor from extract of fermentation of the Peniciliumcyclopium [comp Agricultural and Bioligical Chemistry (Japan), 53, pages544 and 549 (1,969)]. The authors in the article mention that the saidprotease inhibitor does not nevertheless show any inhibition againstpepsin which is a typical acid protease.

Recently, in accompany with a remarkable'advance in the pharmacologicalstudies with regard to ulceration of digestive organs, many attempts are,directed to the aspect of enzymatic action of pepsin in order to solvethe causes of ulceration as well as developments .of ulcers.

It was known that sulfuric acid esters of polysaccharide are effectiveanti-ulcer agent. The pharmacological effects of these substances aresupported mainly by their pepsin inhibitory activity. Unfortunately, itis, however, known that the substantces, when administrated, areaccompanied with injurious side-effect such as prevention ofblood-coagulation.

The first object of the present invention is to provide new pepsininhibitors, namely, the substance 8-H and the substance Me S-PI whichhave no undesirable sideeffect such as that presented by the knownsulfuric acid esters of polysaccharide.

The second object of the present invention is to provide a process forthe production of the said substance SPI by fermentation of a newActimomycetes.

The third object of the present invention is to provide a process forthe production of the substance Me S-Pl by chemical treatment of thesubstance S-PI.

As the result of extensive search over wide range of microorganisms, wehave succeeded in finding out a strain belonging to the genusStreptomyces which is capable of producing a new pepsin inhibitor as ametabolite of said strain. The present invention indeed relys uponthediscoyery and utilization of said microorganism for the production ofsaid new pepsin inhibitor, namely, the substance S-PI.

Surprisingly, we have found that the substance S-Pl exhibits aremarkable activity as pepsin inhibitor, its pepsin inhibitory activityamounting to approximate 10 thousand times of that presented by theknown sulfuric,

acid esters of polysaccharide. The substance S-Pl is an oligopeptidehaving the chemical composition represented by the aforementionedformula, which is quite different from that of the known sulfuric acidesters of polysaccharide, and is free from undesirable side-effect suchas prevention of blood-coagulation.

The substance Me S-Pl also shows pepsin inhibitory effect almostequivalent to that of the substance S-Pl The both substances aretherefore equally utilizable for treatment in safety of patientsuffering from pepticulcer.

The strain utilized in carrying out the process of the present inventionhas been isolated by the present inventors from soil in Japan and namedStreptomyces naniwaensis (EF 44-201).

Specimens of said strain have been deposited to the FermentationResearch Institute of Japan under FER- M-P No. 278, and also to theAmerican Type Culture Collection under the assigned Number ATCC 21689.

The vegetative mycelium of the strain forms many branched mycelia havingdiameter of about 1 micron and forms abundant aerial mycelia.

Well grown aerial mycelium of the strain is frag- I B. Growth of thestrain in various nutrient media at 27C. for 14 days is tabulated in thefollowing Table:

Table I Nutrient medium Status of growth Aerial mycelium Soluble pigmentCzapeck agar trace none asparagine-glucose colorless to light brownishgray agar pale brown glycerol-calcium colorless slightly produced;malate agar grayish white glucose-peptone well grown slightly produced;agar light brownish gray bouillon agar well grown; none pale browntriptone-yeast slightly grown none extract agar starch-casein wellgrown; light brownish gray agar colorless to dark brownStarch-peptonewell grown;

meat CXIIHCI agar pale yellowish light brownish gray light amber noneslightly pale brown none none

none

none

brownish. purple to dark brown tinomycetes. In preparation of a suitablenutrient me dium, there may be employed as essential ingredients,

a nitrogen source such as soybean meal, corn steep li- I Chromogenicaction 2) Cellulose decomposition 3) Thyrosinasc activity 4) Hydrogensulfide formation +++|lll 5) Reduction of nitrate 6) Hydrolysis ofstarch .7) .MQ t assw ty,

8) Coagulation of milk -with strong peptonization 9) Liquefaction ofgelation D) Utilization of carbohydrate l arabinose i 2) raftrnose i 3Xylose 4) galactose 5 rhamnose 6) mannose i 7) lactose 8) maltose 9)fructose l 0) inositol l l salicin l 2) glucose quor, peptone, yeastextract, meat extract, dry yeast, inorganic nitrate, ammonium salt andso on; and a carbon source such as starch hydrolyzate, glucose, molassesand the like. If necessary, there may be used any other suitableinorganic salts such as K l-IPO MgSO FeSO MnSO, etc., and suitableadditives such as antifoaming agent and the like.

The substance S-PI aimed at in the present invention accumulates in thebroth obtained by fermentation of the Streptomyces naniwaensis (EF44-201) in a nutrient medium in accordance with a conventional proceduresuch as a submerged culture, a stationary culture or a surface cultureat a temperature of from about 20C. to about 35C. for about 10-96 hours.

The resulting subtance S-PI may be recovered from the fermented brothadvantageously based on the benefit of its chemical and physicalproperties in accordance with the usual procedure known in the art. Asfor example, the fermented broth may immediately be evaporated todryness; or the broth may first be treated with active carbon to take upthe substance S-PI. In the latter case, the recovered active carbon isthen treated with a water-missible solvent such as methanol to elute thedesired substance. From the eluate, the substance S-PI is recovered byevaporation to dryness.

The active carbon which carries the substance S-PI .may otherwise bewashed with an aqueous alkali solution previous to the step of theaforementioned treat- 6 ered from the fermented broth by means ofprecipita- 0.1-0.2 in methanol-chloroforrn-benaehe (l 1 tion withammonium sulfate. and

The crude substance S-PI, if necessary, may be fur- 0.28-0.32 inbenzene-methanol-acetic acid (80 2O ther purified by conventional columnchromatography 5). on SEPI-IADEXLH-ZO (the registered trade mark), sil-5 2. Properties of the substance Me S-PI ica gel, ion exchange resinsand the like. The substance Me S-PI has a melting point of In additionto the above, a purified substance S-PI 2479-2491:, I V may be obtainedas the precipitate by acidifying the alt S sparingly Soluble in aterthroughout the entire kaline solution of the crude substance at a highconcensphere of pH range; Soluble in m n l n ac tic tration with amineral acid. acid; sparingly soluble in ethanol and butanol. In ben-The production of the substance Me S-PI that constizene, ether,petroleum ether and chloroform, the subtutes the other important aspectof the present invenstance is practically insoluble. tion can beeffected by treating the aforementioned In view of the result of paperelectrophoresis and othcrude, semipurified or purified substance S-PIwith a ers, it is recognized that the substance is a neutral subknownmethyl esterifying agent such as diazomethane stance.

or methanol in the presence of thionyl chloride or dry Within the regionof ultraviolet to visible rays, the hydrogen chloride. The resultingsubstance Me S-Pl substance does not show any particular photomay berecovered by a conventional procedure in a absorption.

form of white powder. Like the free substance S-PI, the Infra-redspectrum of the compound in potassium substance Me S-PI thus obtainedexhibits a marked inbromide tablet is graphically shown in accompanyinghibiting effect against pepsin and other acid proteases. FIG. 2, whereinthe wave lengths in cm unit are given on the abscissa and the percentageabsorptions on the Followings are the physical properties of thesubordinate. In the graph, marked absorptions are obstance S-PI and thesubstance Me S-PI: served at the following wave lengths:.

1. Properties of the substance S-PI 3330, 3080, 1730, 1610-1660,1520-1570, 1440, The substance S-PI begins coloration at about 1380,1370, 1295, 1263, 1225, 1175, 1153, 1072, 2l5C., followed bydecomposition at about 990, 927, 892, 865, 650, 820, 795, 755 and 715.227-230C. It gives no sharp melting point. Specific rotation of thesubstance is from 9l to The substance is-sparingly soluble in water offrom 93 (0 0.1 in methanol). acid to neutral pH range but soluble inalkaline pH In ninhydrine reaction, the substance per se is negarange;soluble in methanol and acetic acid; less soluble tive while itshydrolyzate is positive.

in ethanol and butanol; and practically insoluble in Rydon-Smithreaction of the substance [I-I. N. Rydon benzene, ether, petroleumether, chloroform, carbon and P. W. G. Smith; Nature 169, 922 (1952)] ispositetrachloride, hexane and ethyl acetate. In considertive.

ation of the result of paper electrophoresis and others, Thin layerchromatography of the substance on silica it is recognized that thesubstance S-PI is an acidic subgel GF (the registered trade mark, soldby Merk A.

stance. G. of Germany) gave the following R, values respec- Within theregion of ultraviolet to visible rays, the tively: Y

substance does not show any particular photo- 0.80-0.90 inmethanol-benzene (1 1);

absorption. Photo-adsorptions in the region of infra-red 0.85-0.95 inmethanol-chloroform-benzene (l l rays measured in a potassium bromidetablet are graph- 1); and

ically shown in accompanying FIG. 1 wherein the wave 0.35-0.40 inbenzene-methanol-acetic acid (80 20 lengths in cm unit are given on theabscissa and per- 5).

centage absorption on the ordinate. In the graph, As the results ofinstrumental analyses such as amino marked absorptions are observed atthe following wave acid analysis, gas chromatographic analysis, IR andlengths: NMR and high resolution mass spectrometry, we con- 3320, 3090,2970, 1635, 1520-1555, 1387, 1370, firmed that the substance S-PI hasthe following struc- 1290, 1223, 1175, 1155, 1070 and 8 55.Luraliqgnula; 7V V CH: C13: CH: CH:

CH: CH: CH: CH3 3H (3H ofi ofi (3H2 OH CH: (3H2 OH Specific rotation ofthe substance is from 90 to Pepsin inhibitory effect represented by thesubstance 93 (0 1.0 in methanol). S-PI and the substance Me S-PI weredemonstrated by In ninhydrine reaction, the substance per se is negathefollowing test: tive while its hydrolyzate is positive. To each 2.5 mlof 1% casein aqueous solution (pH Rydon-Smith reaction of the substance[H N. Rydon 2.5), there was added respectively 0.2 ml of'an aqueous andP. W. G. Smith; Nature 169. 922 (1952)] is posisolution of the substanceS-PI or 0.2 mol of an aqueous tive. methanol solution of the substanceMe S-PI, the con- Thin layer chromatography of the substance on silicacentrations of the substances in therespective solutions gel GF (theregistered trade mark, sold by Merk A. varying from one another.

G. of Germany) gave the following R; values respec- To each of theresulting mixtures kept at the tempertively: ature of 37C., was added0.5 ml of a 5% aqueous solu- 0.40-0.50 in methanol-benzene (l 1); tionof pepsin and the whole were incubated at 37C.

To each 1 ml aliquot of the recovered filtrates was added 5 ml of a 0.44molar aqueous solution of sodium carbonate and 1 ml of a phenol reagent(Folins reagent), and the whole were shaken well. After 60 minutes,photoabsorption at 660 mp. wave length of each of the resulting mixtureswas inspected. At the same time, two tests for control were carried outunder the same conditions as those aforementioned with exception thatthe substance S-PI and the substance Me S-PI were eliminated.

In comparison with the control tests, the individual amount of thesubstance S-PI and the substance Me S-Pl required for a 50% diminutionof the pepsin activity were determined, which are then represented bythe term [Pl] as the measure of pepsin inhibitory effect thereof.

. The results thus obtained are:

[Pl] 1.2-1.8 pg for the substance S-PI, and

[Pl] 1.2-1.8 ug for the substance Me S-PI By the aforementioned chemicaland physical as well asbiological characteristics of the substance S-PIand the substance Me S-Pl, they are distinguished from the hithertoknown protease inhibitors and are considered the new compounds.

The following examples serve to illustrate embodiment of the invention.

EXAMPLE 1 under shaking at 27C. for 24 hours in an aqueous nutrientmedium having same composition as the above.

The whole was fermented at 27C. for 24 hours under shaking at 300 r.p.m.and aerating at the rate of 15 liters perminute. The broth was thenfiltered to remove the cells. 1 1 Liters of the filtrate (pH about 5.4)were made to pH 2.0 by adding hydrochloric acid. 2.5% (w/v) of activecarbon were added to the filtrate and the mixture was stirred for about30 minutes to accomplish adsorption of the resultant substance S-PI onthe carbon. The active carbon was recovered by filtration and extractedthree times each with 4 liters of methanol. The combined methanolextracts were subjected to distillationunder reduced pressure to removethe solvent.

The residue was dissolved in 1.1 liters of water. The solution was againtreated with active carbon. The latter was recovered by filtration andwashed two times each with 1 liter of a hot aqueous alkali solution. Inthis treatment, all the colored materials presented as impurity wereremoved almost completely. From the active carbon, the substance S-Plwas eluted with 300 ml of methanol, and the methanol was expelled bydistillation under reduced pressure. There were obtained 2.78 grs. ofthe residue which was dissolved again in methanol. The methanol solutionwas poured onto a column of 3 cm diameter and cm height filled withSEPHADEX LH-20 and eluted with methanol. 150-210 ml of the fractions ofthe eluate were collected and the solvent was removed therefrom bydistillation.

There were obtained 1.58 grs. of the substance S-PI as white amorphouspowder.

EXAMPLE 2 The Streptomyces naniwaensis (EF 44-201) was inoculated to mlof a nutrient medium containing 5% of peptone, 0.1% of sodium chloride,0.1% of dipotassium phosphate, 0.05% of magnesium sulfate, 0.001% offerrous sulfate, 0.0001% of manganese sulfate, 0.0001% of copper sulfateand 0.000l% of zinc sulfate.

The whole was incubated at 27C. for 48 hours under shaking. After theperiod of that time, accumulation of 1.2 mg/ml of the substance S-PI wasfound in the fermented broth.

The broth after filtration was further worked up successively byfractionation with ammonium sulfate, adsorption with active carbon, andcolumn chromatography with SEPHADEX LH-20 and silica gel.

There was obtained 13 mg of the purified substance S-Pl.

Elementary analysis of sodium salt of the substance S-PI gave:

C ,H N O Na (Molecular weight 665.8)

500 mg of substance S-Pl obtained in Example 1 were dissolved in 10 m1of methanol. To the solution, after addition of 15 ml of ether, therewere added dropwise an etheral solution of diazomethane under icecoolinguntil yellow color of the solution was persistent. After completion ofthe reaction, the solvent was removed by distillation under reducedpressure.

The residue was subjected to successive chromatographical treatments onsilica gel and SEPHADEX LH-20. There was obtained 350 mg of thesubstance Me S-PI in a form of white amorphous powder.

The product gave a single spot in thin layer chromatography.

Elementary analysis of the resulting substance gave:

. c ri u o (Molecular weight 657.8)

Calculated 58.42 9.03 10.64 Found 58.59 9.04 10180 EXAMPLE4 Thetreatment with methanol were further repeated ture, at a temperature offrom about 25 C to abou t three times. The resulting residue wasdissolved in a 35C, for from about 10 to 96 hours.

small amount (about ml) of methanol, and any solid 3. A process asclaimed in claim 2 wherein the nutrimaterials were removed byfiltration. The filtrate was ent medium contains a nitrogen sourceselected from subjected to chromatography on silica gel and then on 5 heg p si i g o oy meal, orn ep li- SEPHADEX Ll-l20. There were obtained300 mg of q Q p pt ye eXtfaCt, meat extract, ry y the substance Me S-PIin a form of white amorphous inorganic nitrates, ammonium Salts, andmiXtllfeS powder. thereof, inorganic salts; and a carbon source selectedThe product in thin layer chromatography gave a i from the groupconsisting of starch hydrolyzate, glugle spot. cose molasses, andmistures thereof.

What is claimed i 4.'A process as claimed in claim 1 wherein recovery 1.A process for the preparation of a pepsin i hibit r is effected byabsorption on active carbon followed by having the formula: elution witha water rniscible solvent.

CH3 /CH3 CH: /CH: CH; CH: 053 0Hi CH \(IHI CH CH CH2 OH CH: (EH2 0Hwhich comprises fermenting the Streptomyces naniw- 5. A process as inclaim l wherein recovery is efaensis (EF 44-201: ATCC 21689) in anutrient mefected by precipitation with ammonium sulfate. drum andrecovering said pepsin inhibitor from the fer- 6. A process as in claim4 wherein the recovered submentation broth. stance is purified by columnchromatography.

2. A process as claimed in claim 1 wherein the f r- 7. A process as inclaim 5 wherein the recovered sub mentation is carried out in a nutrientmedium utilizing stan is purified y column chromatography. s b r e PliQ9fl?L Culture urfa e

2. A process as claimed in claim 1 wherein the fermentation is carriedout in a nutrient medium utilizing submerged culture, stationary cultureor surface culture, at a temperature of from about 20*C to about 35*C,for from about 10 to 96 hours.
 3. A process as claimed in claim 2wherein the nutrient medium contains a nitrogen source selected from thegroup consisting of soybean meal, corn steep liquor, peptone, yeastextract, meat extract, dry yeast, inorganic nitrates, ammonium salts,and mixtures thereof, inorganic salts; and a carbon source selected fromthe group consisting of starch hydrolyzate, glucose molasses, andmistures thereof.
 4. A process as claimed in claim 1 wherein recovery iseffected by absorption on active carbon followed by elution with a watermiscible solvent.
 5. A process as in claim 1 wherein recovery iseffected by precipitation with ammonium sulfate.
 6. A process as inclaim 4 wherein the recovered substance is purified by columnchromatography.
 7. A process as in claim 5 wherein the recoveredsubstance is purified by column chromatography.